Apoptosis, or programmed cell death, is a well-defined sequence of events that result in the death of mammalian cells. The process of apoptosis is a normal part of physiology, and a key mechanism in the removal of unwanted cells during various phases of life, for example, fetal development. Upon induction of apoptosis, cells undergo a number of characteristic morphological changes, including cell shrinkage, membrane blebbing, membrane ruffling, loss of cell-cell contact and condensation of nuclear chromatin.
One of the most characteristic events which helps to define apoptosis is the condensation and destruction of nuclear DNA. Following a signal for induction of apoptosis, a variety of nuclear enzymes are activated which cleave DNA at specific points, resulting in production of DNA fragments that are approximately 180-200 base pairs in length. Hence, examination of the DNA of a cell undergoing apoptosis by electrophoresis results in a pattern of xe2x80x9cDNA ladderingxe2x80x9d, which is characteristic of these cells.
As apoptosis is a normal physiological process, dysregulation of the amount of apoptosis occurring in a cell population can be considered as an indicator of existence of a disease state. In certain cancer states, it has been suggested that insufficient apoptosis occurs within the cancerous tumor as a consequence of deletion or mutation of the tumor suppressor gene p53. In contrast, excessive apoptosis is believed to occur in individuals afflicted with Alzheimer""s disease, as evidenced by increased loss of certain neuronal cell types. Increased apoptotic cell death has also been observed in certain T-cell populations in HIV-infected individuals, and in neurons of individuals who have suffered an ischemic event such as a stroke.
Caspase-3 (also known as CPP32, Apopain or Yama) is a 29 kDa cysteine protease. It is a member of a larger family of caspase enzymes, which share sequence homology with one another, including a highly conserved region centered around a cysteine residue believed to be involved in the hydrolysis of the target substrate(s). Included in this larger family is the interleukin-1xcex2 converting enzyme (ICE) and several other mammalian-derived caspases. Much of the understanding of the involvement of caspase-3 in apoptosis has arisen as the result of study of related cysteine proteases expressed by the nematode Caenorhabditis elegans. During normal development of this nematode, 131 of the 1090 cells generated die by apoptosis. Apoptosis of cells during development of C. elegans is vitally dependent upon two enzymes, CED-3 and CED-4, which are cysteine proteases, with CED-3 being highly homologous to both caspase-3 and ICE, including identity of amino acids in the enzyme active site.
Caspase-3 is believed to play a key role in apoptosis. In cells, caspase-3 has been shown to cleave many proteins, including the nuclear enzyme PARP (poly-ADP ribose polymerase), a DNA repair enzyme; U1-70, an enzyme that splices RNA; and DNA-PKCS, an enzyme that repairs double-strand breaks in DNA. As a consequence of the cleavage of these and other proteins by caspase-3, DNA repair is compromised and cells undergo apoptosis. The cleavage of proteins by caspase-3 has been shown to occur at well-defined amino acid sequences in the substrate proteins, in particular at the C-terminal side of a DXXD sequence. Peptide-based inhibitors of caspase-3 capable of blocking the cleavage of protein substrates in assays designed to measure caspase-3 mediated cleavage have been designed. Even though examples of these peptide-based inhibitorsxe2x80x94such as the peptide aldehyde Ac-DEVD-CHOxe2x80x94may inhibit the isolated enzyme, their relative instability to chemical degradation precludes their use as effective inhibitors of caspase-3 in intact cells or in vivo.
Therefore, it would be very desirable to discover other molecules that exhibit similar or better ability to inhibit the cleavage of protein substrates by caspase-3, and possess significantly better physicochemical properties; for example chemical and hydrolytic stability. If discovered, such agents would be expected to be effective at reducing excessive apoptosis, and hence would provide a treatment for diseases characterized by this inappropriate cell death.
One aspect of this invention relates to quinazolines having the general structure I 
wherein R2, R4, R5, R6, R7, R8, R3xe2x80x2, R4xe2x80x2 and R5xe2x80x2 are defined herein.
Another aspect of this invention relates to the use of the above compounds to retard apoptosis in cells and as therapies that are beneficial in the treatment of immune, proliferative and degenerative diseases including, but not limited to, immune deficiency syndromes (such as AIDS), autoimmune diseases, pathogenic infections, cardiovascular and neurological injury, alopecia, aging, cancer, Parkinson""s disease, Alzheimer""s disease, Huntington""s disease, acute and chronic neurodegenerative disorders (e.g. stroke, vascular dementia, head trauma, ALS, neuromuscular disease), myocardial ischemia, cardiomyopathy, macular degeneration, osteoarthritis, diabetes, acute liver failure and spinal cord injury.
A third aspect of this invention relates to pharmaceutical composition containing the above compounds with a pharmaceutically-acceptable carrier or diluent.
The compounds of this invention are quinazolines having the general structure I. 
For structure I, R2 and R4 are, independently, H, acetyl or (C1-C5)alkyl. In another embodiment, R2 and R4 are H.
R5, R6 and R7 are independently selected from H, halogen, (C1-C2)alkyl, halo(C1-C2)alkyl, nitro and cyano. In another embodiment, R6 is selected from halogen, (C1-C2)alkyl, halo(C1-C2)alkyl, nitro and cyano; and R5 and R7 are as above. In another embodiment, R6 is selected from nitro, halogen, xe2x80x94CH3, xe2x80x94CF3 and cyano; and R5 and R7 are independently selected from H, halogen, (C1-C2)alkyl, xe2x80x94CF3, nitro and cyano. In a more specific embodiment, R6 is selected from nitro and halogen.
R8 is selected from H, phenyl, (C1-C6)alkyl, Ri, heterocycle, substituted heterocycle, xe2x80x94(CH2)mC(xe2x95x90O)Nxe2x80x94((CH2)pRg)Rb,xe2x80x94(CH2)mN((CH2)pRg)Rb, xe2x80x94CHxe2x95x90CHxe2x80x94Rc, halogen, xe2x80x94(CH2)mC(xe2x95x90O)(CH2)mRo, xe2x80x94C(xe2x95x90O)Rp, xe2x80x94(CH2)mC(xe2x95x90O)O((CH2)pRg), xe2x80x94(CH2)mN((CH2)pRg)C(xe2x95x90O)Rb, xe2x80x94(CH2)mOC(xe2x95x90O)((CH2)pRg), xe2x80x94CHORdORe, xe2x80x94CH2XRf, xe2x80x94S(xe2x95x90O)2N((CH2)pRg)Rb, xe2x80x94N((CH2)pRg)S(xe2x95x90O)2Rb, xe2x80x94S(xe2x95x90O)2N((CH2)pRg)Rb, xe2x80x94C(xe2x95x90O)H, allyl and 4-hydroxybut-1-en-4-yl. In another embodiment, R8 is selected from H, phenyl, (C1-C6)alkyl, Ri, heterocycle, substituted heterocycle, xe2x80x94(CH2)mC(xe2x95x90O)N((CH2)pRg)Rb, xe2x80x94(CH2)mN((CH2)pRg)Rb, xe2x80x94CHxe2x95x90CHxe2x80x94Rc, halogen, xe2x80x94C(xe2x95x90O)(CH2)mRo, xe2x80x94(CH2)mC(xe2x95x90O)O((CH2)pRg), xe2x80x94(CH2)mN((CH2)pRg)C(xe2x95x90O)Rb, xe2x80x94(CH2)mOC(xe2x95x90O)((CH2)pRg), xe2x80x94CHORdORe, xe2x80x94CH2XRf, xe2x80x94S(xe2x95x90O)2N((CH2)pRg)Rb, xe2x80x94N((CH2)pRg)S(xe2x95x90O)2Rb, xe2x80x94C(xe2x95x90O)H, allyl and 4-hydroxybut-1-en-4-yl. In a more specific embodiment, R8 is xe2x80x94(CH2)mC(xe2x95x90O)N((CH2)pRg)Rb. In another more specific embodiment, R8 is xe2x80x94CHxe2x95x90CHxe2x80x94Rc.
R3xe2x80x2, R4xe2x80x2 and R5xe2x80x2 are independently selected from H, halogen, (C1-C4)alkyl, (C1-C4)alkoxy and halo(C1-C4)alkyl. In another embodiment, R3xe2x80x2, R4xe2x80x2 and R5xe2x80x2 are independently selected from H, halogen and xe2x80x94CF3.
It is important that at least one of R5, R6, R7, R8, R3xe2x80x2 and R5xe2x80x2 is not H; and also that R4xe2x80x2 is not equal to R7.
Rb is independently at each instance H, (C1-C4)alkyl or substituted (C1-C4)alkyl. In another embodiment, Rb is H, xe2x80x94CH3 or xe2x80x94CH2CH3.
Rc is independently at each instance selected from H, phenyl, Ri, heterocycle, substituted heterocycle, xe2x80x94CO2Rb, xe2x80x94C(xe2x95x90O)NRbRb, xe2x80x94S(xe2x95x90O)nxe2x80x94Rf, 2-hydroxyisopropyl and cyano. In another embodiment, Rc is selected from phenyl, Ri, heterocycle, xe2x80x94CO2Rb, xe2x80x94C(xe2x95x90O)NRbRb, xe2x80x94OC(xe2x95x90O)Rb, xe2x80x94NRbC(xe2x95x90O)Rb, xe2x80x94S(xe2x95x90O)nxe2x80x94Rf, 2-hydroxyisopropyl and cyano.
Rd and Rc are independently at each instance (C1-C4)alkyl; or Rd and Re together are xe2x80x94CH2CH2xe2x80x94 or xe2x80x94CH2CH2CH2xe2x80x94, giving the following ring substituents: 
Rf is independently at each instance (C1-C4)alkyl, vinyl, xe2x80x94CH2CO2Rb, phenyl or benzyl.
Rg is independently at each instance selected from (C1-C10)alkyl, substituted (C1-C10)alkyl, phenyl, Ri, heterocycle, substituted heterocycle, xe2x80x94ORb, xe2x80x94NRbRb, xe2x80x94NRjRo, xe2x80x94N(Rj)SO2Rj, xe2x80x94CO2Rb, xe2x80x94C(xe2x95x90O)NRjRj, xe2x80x94SO2phenyl and 2-oxopyrrolid-1-yl; or Rg and Rb together form xe2x80x94CH2CH2N(Rj)CH2CH2xe2x80x94, xe2x80x94(CH2)4xe2x80x94, xe2x80x94CH(Rh)CH2CH2CH2xe2x80x94, or xe2x80x94CH2CH2OCH2CH2xe2x80x94, forming the following rings: 
respectively. In another embodiment, Rg is selected from (C1-C10)alkyl, substituted (C1-C10)alkyl, phenyl, Ri, heterocycle, substituted heterocycle, xe2x80x94ORb, xe2x80x94NRbRb, xe2x80x94CO2Rb and 2-oxopyrrolid-1-yl. In another embodiment, Rg is selected from (C1-C6)alkyl, phenyl, Ri and heterocycle. Some specific examples of Rg include, but are not limited to, isopropyl, phenyl, 4-fluorophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2-chlorophenyl, 4-chlorophenyl, 2,4-dichlorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl, 3-bromophenyl, 4-bromophenyl, 4-(trifluoromethyl)phenyl, 2-(trifluoromethyl)phenyl, 4-(trifluoromethoxy)phenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 4-methoxyphenyl, 3-iodophenyl and 3-fluoro-5-(trifluoromethyl)phenyl.
Rh is independently at each instance xe2x80x94CO2Rf or xe2x80x94CH2O-phenyl.
Ri is independently at each instance phenyl, containing one, two or three substituents selected from halogen, (C1-C6)alkyl, xe2x80x94ORj, xe2x80x94O(substituted phenyl) xe2x80x94NRjRj, halo(C1-C6)alkyl, halo(C1-C4)alkoxy, nitro, xe2x80x94C(xe2x95x90O)Rj, xe2x80x94C(xe2x95x90O)(substituted phenyl), xe2x80x94(CH2)mC(xe2x95x90O)NRjRk, xe2x80x94(CH2)mC(xe2x95x90O)N(Rj)SO2((C1-C6)alkyl), xe2x80x94(CH2)mC(xe2x95x90O)NRj(substituted phenyl), xe2x80x94(CH2)nCO2Rj, xe2x80x94OC(xe2x95x90O)Rj, xe2x80x94N(Rj)C(xe2x95x90O)Rj, xe2x80x94NRjC(xe2x95x90O)xe2x80x94halo(C1-C4)alkoxy, xe2x80x94C(xe2x95x90O)NRjRj, xe2x80x94NRjS(xe2x95x90O)2(C1-C4)alkyl, xe2x80x94SOn(C1-C6)alkyl, xe2x80x94SOn(halogen), xe2x80x94SOm(CH2)phenyl, xe2x80x94SO2NRjRj, xe2x80x94SO2NRjRk, xe2x80x94SO2NRj(substituted (C1-C6)alkyl), xe2x80x94SO2(CH2)nRo, xe2x80x94SO2N(Rj)(CH2)Ro, xe2x80x94SOn(halo(C1-C3)alkyl), xe2x80x94SOn(pyrrolidin-1-yl substituted in the 2 position by Rn), xe2x80x94CN, xe2x80x94SCN, phenyl, heterocycle and benzyl. In another embodiment, Ri is phenyl, containing one, two or three substituents selected from halogen, (C1-C6)alkyl, ORj, xe2x80x94NRjRj, halo(C1-C6)alkyl, halo(C1-C4)alkoxy, nitro, xe2x80x94CO2Rj, xe2x80x94OC(xe2x95x90O)Rj, xe2x80x94N(Rj)C(xe2x95x90O)Rj, xe2x80x94NRjC(xe2x95x90O)xe2x80x94halo(C1-C4)alkoxy, xe2x80x94C(xe2x95x90O)NRjRj, xe2x80x94NRjS(xe2x95x90O)2(C1-C4)alkyl, xe2x80x94SOn(C1-C6)alkyl, xe2x80x94SOn(halogen), xe2x80x94SOnphenyl, xe2x80x94SO2NRjRj, phenyl and benzyl.
Rj is independently at each instance H or (C1-C6)alkyl.
Rk is independently at each instance xe2x80x94(CH2)nCH2OCH2Rb, xe2x80x94C(xe2x95x90O)NRjRj or xe2x80x94C(xe2x95x90O)Rj.
Rm is independently at each instance heterocycle, containing one or two substituents selected from halogen, (C1-C6)alkyl, xe2x80x94ORj, xe2x80x94O(substituted phenyl)-NRjRj, halo(C1-C6)alkyl, halo(C1-C4)alkoxy, nitro, xe2x80x94C(xe2x95x90O)Rj, xe2x80x94C(xe2x95x90O)(substituted phenyl), xe2x80x94(CH2)mC(xe2x95x90O)NRjRk, xe2x80x94(CH2)mC(xe2x95x90O)N(Rj)SO2((C1-C6)alkyl), xe2x80x94(CH2)mC(xe2x95x90O)NRj(substituted phenyl), xe2x80x94(CH2)nCO2Rj, xe2x80x94OC(xe2x95x90O)Rj, xe2x80x94N(Rj)C(xe2x95x90O)Rj, xe2x80x94NRjC(xe2x95x90O)xe2x80x94halo(C1-C4)alkoxy, xe2x80x94C(xe2x95x90O)NRjRj, xe2x80x94NRjS(xe2x95x90O)2(C1-C4)alkyl, xe2x80x94SOn(C1-C6)alkyl, xe2x80x94SOn(C1-C6)alkyl, xe2x80x94SOn(halogen), xe2x80x94SOm(CH2)nphenyl, SO2NRjRj, xe2x80x94SO2NRjRk, xe2x80x94SO2NRj(substituted (C1-C6)alkyl), xe2x80x94SO2(CH2)nRo, xe2x80x94SO2N(Rj)(CH2)nRo, xe2x80x94SOn(halo(C1-C3)alkyl), xe2x80x94SOn(pyrrolidin-1-yl substituted in the 2 position by Rn), xe2x80x94CN, xe2x80x94SCN, phenyl, heterocycle and benzyl.
Rn is independently at each instance xe2x80x94C(xe2x95x90O)Rj, xe2x80x94CH2ORj or xe2x80x94C(xe2x95x90O)NRjRj.
Ro is independently at each instance phenyl, substituted phenyl, heterocycle or substituted heterocycle.
Rp is independently at each instance heterocycle, containing one or two substituents selected from substituted phenyl, heterocycle, phenyl, benzyl, xe2x80x94SOnRo or SO2NRjRj.
m is independently at each instance 0, 1, 2 or 3. In another embodiment, m is 0, 1 or 2. In another embodiment, m is 0.
n is independently at each instance 0, 1 or 2.
p is independently at each instance 0, 1, 2, 3, 4, 5, 6 or 7. In another embodiment, p is 0, 1, 2, 3 or 4. In another embodiment, p is 0 or 1. In another embodiment, p is 1.
X is independently at each instance S, O or N.
Specific compounds within the scope of the invention also include, but are not limited to the examples shown in this specification.
(CY-CZ)alkyl, unless otherwise specified, means an alkyl chain containing a minimum Y total carbon atoms and a maximum Z total carbon atoms. These alkyl chains may be branched or unbranched, cyclic, acyclic or a combination of cyclic and acyclic. For example, the following substituents would be included in the general description xe2x80x9c(C4-C7)alkylxe2x80x9d: 
Substituted (CY-CZ)alkyl means (CY-CZ)alkyl, as defined above, substituted by one, two or three substituents selected from halogen, hydroxy, amino, (C1-C6)alkoxy, halo(C1-C4)alkoxy, xe2x80x94CO2H, xe2x80x94CO2(C1-C4)alkyl, xe2x80x94OC(xe2x95x90O)xe2x80x94(C1-C6)alkyl and benzyl; preferably containing one or two substituents selected from halogen, trifluoromethyl, (C1-C4)alkoxy, and (C1-C6)alkyl; and more preferably selected from hydroxy, methoxy and methyl. Examples of substituted (CY-CZ)alkyls include, but are not limited to, 3-carboxycyclohexyl and 2,2-dihydroxymethylbutyl. xe2x80x9cSubstituted phenylxe2x80x9d means a phenyl group, containing one, two or three substituents selected from halogen, hydroxy, amino, (C1-C6)alkoxy, halo(C1-C4)alkoxy, (C1-C6)alkyl, halo(C1-C6)alkyl, nitro, xe2x80x94CO2H, xe2x80x94CO2(C1-C6)alkyl, xe2x80x94OC(xe2x95x90O)(C1-C6)alkyl, xe2x80x94NHC(xe2x95x90O)(C1-C4)alkyl, xe2x80x94N((C1-C6)alkyl)C(xe2x95x90O)(C1-C4)alkyl, xe2x80x94NHC(xe2x95x90O)xe2x80x94halo(C1-C6)alkoxy, xe2x80x94N((C1xe2x80x94C6)alkylC(xe2x95x90O)xe2x80x94halo(C1-C4)alkoxy, xe2x80x94C(xe2x95x90O)N((C1-C6)alkyl)((C1-C6)alkyl), xe2x80x94C(xe2x95x90O)N((C1-C6)alkyl)H, xe2x80x94C(xe2x95x90O)NH2, xe2x80x94NHS(xe2x95x90O)2(C1-C4)alkyl, xe2x80x94N((C1-C6)alkyl)SO2(C1-C6)alkyl, xe2x80x94SOn(C1-C6)alkyl, xe2x80x94SOn(halogen), xe2x80x94SOnphenyl, xe2x80x94SO2N((C1-C6)alkyl)(C1-C6)alkyl), xe2x80x94SO2N((C1-C6)alkyl)H, xe2x80x94SO2NH2, phenyl and benzyl. Preferably, substituted phenyls contain one or two substituents selected from halogen, trifluoromethyl, (C1-C4)alkoxy, and (C1-C6)alkyl; and more preferably, selected from chlorine, fluorine, methoxy, and methyl.
Additionally, a substituted phenyl may be substituted by a functional group containing a second substituted phenyl such as -(substituted phenyl), xe2x80x94C(xe2x95x90O)(substituted phenyl), xe2x80x94SO2(substituted phenyl) and xe2x80x94O(substituted phenyl); the second (terminal) substituted phenyl may contain one, two or three substituents selected from halogen, hydroxy, amino, (C1-C6)alkoxy, halo(C1-C4)alkoxy, (C1-C6)alkyl, halo(C1-C6)alkyl, nitro, xe2x80x94CO2(C1-C6)alkyl, xe2x80x94OC(xe2x95x90O)xe2x80x94(C1-C6)alkyl, xe2x80x94NC(xe2x95x90O)xe2x80x94(C1-C4)alkyl, xe2x80x94NC(xe2x95x90O)xe2x80x94halo(C1-C4)alkoxy, xe2x80x94C(xe2x95x90O)N((C1-C6)alkyl)((C1-C6)alkyl or H), xe2x80x94NS(xe2x95x90O)2(C1-C4)alkyl, xe2x80x94SO2(C1-C6)alkyl, xe2x80x94SO2(halogen), xe2x80x94SO2phenyl, xe2x80x94SO2N((C1-C6)alkyl)((C1-C6)alkyl, phenyl and benzyl.
xe2x80x9cHeterocyclexe2x80x9d means a five- or six-membered ring, saturated or unsaturated, containing one, two or three heteroatoms selected from N, O and S, with the remainder of the ring being made up of carbon atoms, wherein the heteroatomic ring may be fused with a phenyl ring to form a bicyclic heterocycle; preferably, pyridyl, furyl, indolyl, indazolyl, morpholino, thiazolyl, imidazolyl or pyridizinyl.
xe2x80x9cSubstituted heterocyclexe2x80x9d in this application means a heterocycle, as defined above, that contains one or two substituents selected from halogen, trifluoromethyl, (C1-C4)alkoxy and (C1-C6)alkyl.
xe2x80x9cHalo(C1-C4)alkylxe2x80x9d means (C1-C4)alkyl substituted by one, two or three halogen atoms.
xe2x80x9cHalo(C1-C4)alkoxyxe2x80x9d means xe2x80x94Oxe2x80x94(C1-C4)alkyl substituted by one, two or three halogen atoms.
Some individual compounds within the scope of this invention may contain double bonds. Representations of double bonds in this invention are meant to include both the E and the Z isomer of the double bond.
Additionally, some species within the scope of this invention may contain one or more asymmetric centers. This invention includes the use of any of the optically pure stereoisomers as well as any combination of stereoisomers.
The compounds of the present invention are capable of forming salts with various inorganic and organic acids and bases and such salts are also within the scope of this invention. Examples of such acid addition salts include acetate, adipate, ascorbate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, citrate, ethanesulfonate, fumarate, glutamate, glycolate, hemisulfate, 2-hydroxyethylsulfonate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, hydroxymaleate, lactate, malate, maleate, methanesulfonate, 2-naphthalenesulfonate, nitrate, oxalate, pamoate, persulfate, phenylacetate, phosphate, picrate, pivalate, propionate, salicylate, stearate, succinate, sulfamate, sulfanilate, sulfate, tartrate, tosylate (p-toluenesulfonate), and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium, lithium and potassium salts, alkaline earth metal salts such as aluminum, calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, ornithine, and so forth. Also, basic nitrogen-containing groups may be quaternized with such agents as: lower alkyl halides, such as methyl, ethyl, propyl, and butyl halides; dialkyl sulfates like dimethyl, diethyl, dibutyl; diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl halides; aralkyl halides like benzyl bromide and others. Non-toxic physiologically-acceptable salts are preferred, although other salts are also useful, such as in isolating or purifying the product.
The salts may be formed by conventional means, such as by reacting the free base form of the product with one or more equivalents of the appropriate acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water, which is removed in vacuo or by freeze drying or by exchanging the anions of an existing salt for another anion on a suitable ion-exchange resin.
For oral use of a compound according to this invention, the selected compound may be administered, for example, in the form of tablets or capsules, or as an aqueous solution or suspension. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch, and lubricating agents, such as magnesium stearate, are commonly added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents may be added. For intramuscular, intraperitoneal, subcutaneous and intravenous use, sterile solutions of the active ingredient are usually prepared, and the pH of the solutions should be suitably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled in order to render the preparation isotonic.
In addition to the novel compounds described above, this invention also relates to a method of treating a mammalian disease selected from cell apoptosis, immune deficiency syndromes, autoimmune diseases, pathogenic infections, cardiovascular and neurological injury, alopecia, aging, cancer, Parkinson""s disease, Alzheimer""s disease, Huntington""s disease, acute and chronic neurodegenerative disorders, stroke, vascular dementia, head trauma, ALS, neuromuscular disease, myocardial ischemia, cardiomyopathy, macular degeneration, osteoarthritis, diabetes, acute liver failure and spinal cord injury, comprising the step of administering a therapeutically-effective amount of a compound as described above.
The present invention also encompasses a pharmaceutical composition useful in retarding apoptosis in cells and as therapies that are beneficial in the treatment of immune, proliferative and degenerative diseases including, but not limited to, immune deficiency syndromes (such as AIDS), autoimmune diseases, pathogenic infections, cardiovascular and neurological injury, alopecia, aging, cancer, Parkinson""s disease, Alzheimer""s disease, Huntington""s disease, acute and chronic neurodegenerative disorders (e.g. stroke, vascular dementia, head trauma, ALS, neuromuscular disease), myocardial ischemia, cardiomyopathy, macular degeneration, osteoarthritis, diabetes, acute liver failure and spinal cord injury, comprising the administration of a therapeutically-effective amount of the compounds of this invention, with or without pharmaceutically-acceptable carriers or diluents. Suitable compositions of this invention include aqueous solutions comprising compounds of this invention and pharmaceutically-acceptable carriers, e.g., saline, at a pH level, e.g., 7.4. The solutions may be introduced into a patient""s intramuscular blood-stream by local bolus injection.
When a compound according to this invention is administered into a human subject, the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, and response of the individual patient, as well as the severity of the patient""s symptoms. In one exemplary application, a suitable amount of compound is administered to a mammal undergoing treatment for neurodegeneration. Administration occurs in an amount between about 0.1 mg/kg of body weight to about 30 mg/kg of body weight per day, preferably of between 0.1 mg/kg of body weight to about 3 mg/kg of body weight per day.
Preparation of Recombinant Human Caspase-3
A full-length human caspase-3 cDNA was isolated from a human monocyte library by PCR and cloned into pUC18. Following sequencing, individual p17 and p12 subunits were subcloned by PCR into pET21a(+) plasmids, re-sequenced and then transformed into BL21 (DE3) E. Coli. The cells were then lysed, inclusion bodies collected, washed and solubilized. The solubilized subunits were mixed in equimolar ratio to achieved assembly of mature enzyme, which was dialyzed into a reaction buffer. Of the total mature enzyme, approximately 10% was catalytically active. Caspase-3 was purified to homogeneity by Q sepharose chromatography and analyzed with the fluorogenic substrate Ac-DEVD-AMC (Acetyl-aspartyl glutamyl valyl aspartyl-amino methyl coumarin), yielding the following kinetic parameters: Km=20xc2x11.0 xcexcM; kcat=76xc2x11 Sxe2x88x921; kcat/Km=3.8xc3x97106 Mxe2x88x921*Sxe2x88x921; Vmax 10.1xc2x10.2 xcexcM AMC/min/xcexcg protein.
Caspase-3 Inhibition Assay
The recombinant human caspase-3 was simultaneously co-incubated with substrate and increasing concentrations of tested compound (1xc3x9710xe2x88x929 Mxe2x88x925xc3x9710xe2x88x925 M), in a 96-well plate format. The substrate (final concentration of 10 xcexcM) and the enzyme (final concentration of 0.1 xcexcg/mL) were diluted in assay buffer containing 150 mM NaCl, 50 mM HEPES, 5 mM EDTA and 1 mM DTT, pH 7.0. Test compounds were dissolved in DMSO. Caspase-3 activity was determined from the initial rate of Ac-DEVD-AMC hydrolysis by following the accumulation of a fluorogenic product AMC over time. AMC formation was detected from increase in sample fluorescence (xcexex=360 nm, xcexem=460 nm) using a 1420 Victor multilabel plate counter (Wallac), acquiring sample reading every 2 minutes for one hour.
Data Analysis
Data are collected in Excel software files, calculated and formatted for analysis by Prizm software (GraphPad). Substrate concentrations vs. initial velocities were analyzed by nonlinear regression fit to the Michaelis-Menten equation to derive basic kinetic parameters (e.g. Km, Vmax and Kcat). For reversible inhibitors, velocities of the reaction were determined by linear regression analysis. Kiapp was obtained using Dixon plot, which is a linear regression analysis of inhibitor concentrations vs. 1/velocities (V0). Ki values were calculated from Dixon plots utilizing the equation Ki=Kiapp/(1+[S]/Km). For slow binding inhibitors, the association rate constants (kon) were determined by fitting the data into the established pseudo first-order exponential rate equation (kobs). A plot of inhibitor concentration vs. kobs yielded the kinetic constants kon and koff and an apparent affinity constant Kixe2x80x2 was calculated by solving for koff/kon. The reference inhibitor Ac-DEVD-CHO demonstrated a Kixe2x80x2=6.1xc2x10.3 nM (n=100).
Apoptosis Assay (PC12 Cells)
Preparation of Cells for Assay
Rat pheochromocytoma (PC12) cells were obtained from American Type Tissue Collection (Cat. #CRL-1721) and grown in RPMI-1640 media supplemented with 15% fetal bovine serum (FBS) and 1% L-glutamine. RPMI-1640 and L-glutamine were obtained from Gibco, FBS was from Hyclone. Cells were plated on 100 mm Collagen I plates (Becton Dickinson) at a density of approximately 1xc3x97106xe2x88x922xc3x97106, and passed every other day at a ratio of 1:10.
The PC12 cells were plated onto 96 well plates at a density of approximately 1xc3x97104 cells/well and were then differentiated for 9-14 days in RPMI-1640 media supplemented with 1% FBS+50 ng/mL Nerve Growth Factor (NGF). NGF withdrawal was accomplished by washing the cells once with NGF-free medium followed by incubation in NGF-free medium containing rabbit antibody against 2.5 S NGF (anti-NGF Ab) at a 1:400 dilution. NGF (2.5 S, Cat. #N6009) and an anti-NGF antibody (Cat. #N6655) were purchased from Sigma.
Apoptosis Assay
PC12 cells plated in 96 well plates with NGF-free media were incubated in the presence or absence of compound for 3 hours in a 5% CO2, 37xc2x0 C. incubator. After 3 hours, the supernatant was removed and the cells were resuspended in 200 xcexcL of Lysis buffer (#5) provided with the Cell Death Detection Assay kits purchased from Boehringer (Cat. #1774425). The cells were incubated at room temperature for 30 min. Next, the plate was spun for 10 min at 200xc3x97g (xcx9c1100 rpm). The cell lysate (20 xcexcL) was transferred into each well into a streptavidin-coated microtiter plate (provided with the Boehringer kit). The Immunoreagent mixture from the kit (80 xcexcL) was then added to each well. Adhesive foils were used to cover the plates and they were incubated overnight in the refrigerator. The wells were then washed 3 times with ca. 250 xcexcL of Incubation Buffer (#4) from the kit. Substrate Solution (#6) (100 xcexcL) from the kit was added to each well and the plate was incubated at room temperature for about 20 min or until color development occurred. The calorimetric assay was quantitated at 405 nm, with a reference wavelength at 495 nm, in a plate reader. A standard inhibitor (Boc-Asp(OCH3)xe2x80x94CH2F, obtained from Enzyme Systems Products (Cat. #FK-011)), was employed to validate results from each plate.